Activation of T-cells depends on numerous interactions of surface receptors and cytoplasmic proteins. Cell adhesion and recognition of foreign peptides presented by MHC molecules is mediated by extracellular portions of T-cell receptors, such as the adhesion domains of CD2 and the Fv fragment of the alpha-beta heterodimer (Ti) of the T-cell receptor of antigens (TCR). During the previous granting period we have characterized structures of several extracellular portions of such receptors, such as the adhesion domains of CD2, CD58 and a Fv fragment of the class II-specific alpha-beta-TCR Dl0, and we have elucidated the role of glycans in the adhesion domains of CD2 and CD58. Finally, we determined the structures of the CD2/CD58 complex and a complex between the Dl0 Fv fragment and a peptide/MHC Class II complex. The proposed continuation of this research is focused on structural and functional characterization of CD3 components of the TCR, the signaling components of the TCR, and on proteins that mediate signaling via the cytoplasmic tail of CD2. We will pursued via three specific aims: 1. Structure and interactions of the heterodimer: We will solve the structure of a recently engineered correctly folded single chain epsilon-gamma-CD3 construct using NMR spectroscopy. Based on the structure we will investigate interactions between the epsilon-gamma-CD3 heterodimer and the alpha-beta-TCR. 2. Structure and interactions of the epsilon-delta-CD3 heterodimer: Using similar strategies we will engineer single-chain constructs of the epsilon-delta-CD3 heterodimer and study its interaction with the alpha-beta-TCR and CD4. 3. Structural and functional studies of CD2-BP2: We will solve the structure of the complex of the GYF domain of CD2-BP2 with a fragment of the CD2 tail. We will also study larger fragments of or full-length CD2-BP2 and investigate their interactions with CD2 or potential other factors. This research will provide new insights into the mechanism of T-cell activation at the receptor-cytoplasm border.